Archives of Insect Biochemistry and Physiology 18:285-300 (1991)

Bacteria-Induced Protein P4 (Hemolin) From Manduca sexta: A Member of the Immunoglobulin Superfamily Which Can

Inhibit Hemocyte Aggregation Noma E. Ladendorff and Michael R. Kanost Department of Biochemistry and Center fir Insect Science, University of Arizona, Tucson, Arizona The synthesis of a number of hemolymph proteins i s induced in insects i n response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of M, = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NHp-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and i s composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence i s 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hernocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects. Key words: hemolymph proteins, insect immunity, tobacco hornworm

Acknowled ments: We thank Wallace Clark for amino acid sequencing, Dr. RikVanAntwerpen for help w i t f immunofluorescence staining, Drs. Gail Burd and Leslie Tolbert for use of microscopes, Dr. Peter Dunn for providing a cDNA library, and Dr. Michael Wells for the use of laboratory facilities. This research was supported by the Center for Insect Science (National Science Foundation Grant DIR-8720082) and by National Institutes of Health Grants GM41247 and HL39116. Received April 12,1991; accepted August 9,1991. Address reprint requests to Michael R. Kanost, Department of Biochemistry, Kansas State University, Manhattan, KS 66506. The nucleotide sequence reported in this paper has been submitted to the CenBanklEMBL Data Bank with accession number M64346.

0 1991 Wiley-Liss, Inc.

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INTRODUCTION

Insects are able to respond to bacterial infections and to invasion of the hemocoel by eukaryotic parasites with a variety of cellular and humoral responses [for reviews, see 1-41. The cellular responses of hemocytes include phagocytosis, nodule formation, and encapsulation,which efficiently clear most foreign objects from the hemocoel. The humoral responses of insects are relatively well understood only for bacterial infections. Within hours after injection of bacteria, the fat body begins to synthesize and secrete a battery of antibacterial hemolymph proteins. These have been investigated most thoroughly in two lepidopteran species, Hyalophora cecropia and Manducu sexta, and in three dipteran species, Sarcophuga peregrina, Phorrnia terranavae, and Drosophila melunogaster. Injection of living bacteria or killed bacterial “vaccines” induces synthesis of the bacteriolytic enzyme, lysozyme, and several types of antibacterial peptides including cecropins, attacins, diptericins, and defensins [4] which act by damaging bacterial cell membranes. In all cases that have been studied so far, the increased synthesis of these antibacterial proteins results from elevated levels of their mRNAs, predominantly in the fat body [5-81. The presence of antibacterial proteins in the hemolymph after a vaccination with killed bacteria corresponds with a protected state which lasts for a period of several days, during which normally lethal doses of pathogenic bacteria can be survived [l]. The mechanisms by which insect cells recognize nonself, triggering the defensive responses described above, are not understood. The induction of antibacterial proteins is nonspecific; all species of bacteria tested induce the same set of proteins [9,10]. Insect hemocytes phagocytose or adhere to nearly any foreign object, whether biotic or abiotic. There is some evidence that charge or hydrophobicity of a surface may influence the extent of hemocyte responses [3], and lectins in hemolymph have been proposed as possible recognition molecules [2,11]. However, the biochemical interactions which result in binding of hemocytes to foreign objects, and to each other during nodule formation and encapsulation are still to be discovered. Antibodies have not been found in any invertebrate [ll],although proteins which are members of the Ig“ gene superfamily are known to function as cell adhesion molecules during development of the insect nervous system [ 12-14]. It appears that the Ig gene superfamily existed prior to the evolutionary divergence of vertebrates and invertebrates, but that rearranging genes required for generating the diversity of antibodies and T cell receptors have evolved only in vertebrates [ll].It remains possible, however, that nonrearrangng genes from the Ig superfamily, such as those encoding leukocyte adhesion molecules [15-171, could have a role in immune surveillance in invertebrates. In fact, Sun et al. [18] have recently shown that bacteria-induced hemolymph protein P4 (hemolin) from H. cecropia is a member of the Ig superfamily. In this study we have isolated and sequenced a cDNA clone for P4 from larvae of M . sexta. The protein contains four C2 type Ig domains. This glycoprotein of M, = 48,000 is the major bacteria-induced protein in M. sexta [19] and is homologous with protein P4 of N.cecropia [20]. M . sexta P4 is 60% identical in amino acid sequence with P4 (hemolin) from H. cecropia [MI. Although *Abbreviations used: Ig = immunoglobulin, M,

=

relative molecular weight.

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its function in vivo has not yet been determined, M . sexta P4 protein binds to bacterial and hemocyte surfaces and can inhibit hemocyte aggregation. MATERIALS AND METHODS cDNA Libraries and Library Screening A rabbit antiserum to M . sexta protein P4 [19] was used to screen a cDNA library in lambda gtll prepared from fat body RNA of larvae injected with bacteria (providedby P.E. Dunn, Purdue University, West Lafayette, IN). Screening was performed as described by Cole et al. [21]. Six positive clones were isolated, and all contained inserts of approximately 1,100 bp. Inserts from two of these clones (P4-1 and P4-2) were subcloned into the Eco RI site of plasmid pTZ18U. These clones were found to be incomplete, and were used to screen a new cDNA library constructed as follows. RNA was extracted [22] from fat bodies of 10 day 3 fifth instar M . sextu larvae which had each been injected 24 h previously with 100 pg of Micrococcus Zysodeiktikus cells (Sigma, St. Louis, MO) [19]. Poly(A)+ RNA was prepared by binding to an oligo(dT) column (Poly A Quik, Stratagene, La JolIa, CA). cDNA was synthesized from 5 pg poly(A) RNA and directionally cloned into the Sal I and Not I sites of plasmid pSPORT (BRL, Gaithersburg, MD). The resulting plasmids were used to transform Escherichiu coli strain DH5a, resulting in a library containing 120,000 recombinants. Five thousand clones from the library were screened by colony hybridization [23] using the insert from cDNA clone P4-1 labeled with 32Pas a probe. Ten positive clones were isolated and analyzed by restriction mapping and partial or complete DNA sequencing.

+

DNA Sequencing Unidirectional deletion clones for DNA sequencing were produced by digestion with exonuclease I11 [24]. Double stranded DNA or single stranded DNA generated from the f l origin of replication of the plasmids was sequenced by the dideoxy chain termination method [25] using modified T7 DNA polymerase (United States Biochemical, Cleveland, OH) [26]. The sequences of both DNA strands were determined. Protein Sequencing Protein P4 was isolated as described previously [19], and NH2-terminal sequence was determined by Edman degradation using an Applied Biosystems477A protein sequencer with on line phenylthiohydantoin amino acid analyzer 120A. Northern Analysis Day 2 fifth instar M . sexfa larvae and day 1 adult males were injected with 100 pg M. lysodeikficus in 10 (1.1 water or with 10 PI sterile, filtered (0.22 pm) water as a control, and fat body RNA was isolated as described above at different times after injection. Samples of total RNA (10 pg per lane) were fractionated by electrophoresis in an agarose gel containing formaldehyde [23] and transferred to Nylon membrane (Hybond N, Amersham, Arlington Heights, IL). Hybridization with the cDNA insert from clone P4-1 labeled with 32Pwas carried out as described by Cole et al. [21].

Hemocyte Aggregation Experiments Hernolymph was collected into chilled polypropylene tubes from day 2 fifth instar M . sextu larvae by dorsal vessel puncture [27]. Samples of hemolymph

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(20 pl, approximately lo4 hemocytes) were added to wells of a 96 well plate (#3072 Falcon, Becton Dickinson, Lincoln Park, NJ) containing 20 pl50 mM MOPS, 50 mM glutathione, pH 7.0, and either 10 pg of P4 protein or 10 k g of ovalbumin (Sigma) in 20 pl 50 mM MOPS, pH 7.0. The plate was incubated 3 h at 24°C with shaking at 120 rpm, and cells were then viewed by phase contrast microscopy. Immunofluorescence Staining

Hemolymph (approximately 0.5 mlhnsect) was collected from chilled day 2 M. sextu larvae by dorsal vessel puncture [27] and diluted in a sterile polypropylene tube in 1 ml cold Excell-400 insect cell culture medium (J.R. Scientific, Woodland, CA). The cells were pelleted by centrifugation (5008, 10 min, room temperature), resuspended in 50 pl Excell-400 medium, transferred to glass slides, and allowed to attach for 30 min. The medium was removed and replaced with 50 p1 Excell-400 medium containing 10 kg P4 protein or with medium alone and incubated 30 min. The cells were washed with 3 ml Excell-400 medium and then covered with 50 k1 medium containing P4 antiserum (1:1,000 dilution) or medium containing preimmune serum from the same rabbit (1:1,000) and incubated for 1 h. After washing with 3 ml medium the cells were covered with 50 p1medium containing goat anti-rabbit IgG labeled with rhodamine (Boehringer Mannheim, Indianapolis, IN, 1:50 dilution) and incubated 1 h. The cells were counterstained for 5 min with Hoechst dye #33258 (Sigma) and then washed with 3 ml medium. Association of Hemolymph Proteins With Bacteria

Four day 2 fifth instar larvae were injected with 100 pg M. lysodeikticus and four larvae were left untreated as controls. After 24 h, hemolymph was collected into tubes containing a few crystals of 1-phenyl-2-thioureaand pooled from each group. After removal of hemocytes by centrifugation, hemolymph proteins were desalted over a PDlO column (Pharmacia, Piscataway, NJ) with 10 mM Tris-HC1, pH 8.2. The protein samples were then each split into two equal parts, and to one part bovine serum albumin (Fraction V, Calbiochem, La Jolla, CA) was added to a final concentration of 10 rng/ml. A suspension of late log phase E . coli (strain JM101)was prepared as described by Sun et al. [18]. To test binding of hemolymph proteins to the bacterial surface, 0.2 ml of bacterial suspension was mixed with 0.2 ml of hemolymph proteins and incubated for 1 min at room temperature. The bacteria were then pelleted by centrifugation (lO,OOOg, 2 min), washed twice with 1 ml of water, and suspended in 200 p,1 of 0.5 M ammonium formate, pH 6.4 [18]. The bacteria were removed by centrifugation, and the eluted proteins were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis followed by staining with Coomassie blue or immunoblotting [19]. RESULTS Isolation and Sequencing of P4 cDNA Clones

Antiserum to bacteria-induced protein P4 from M. sextu [19] was used to screen a lambda gtll expression library made from fat body RNA of larvae

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previously injected with bacteria. Six positive clones were isolated, and all contained cDNA inserts of approximately 1,100 bp. Partial sequencing of these showed that they appeared identical, with all ending at the same position at the 5’ end with an Eco RI site that was not preceded by a linker sequence. It seemed that during construction of the library the Eco RI sites were not adequately protected before removing excess linkers, and an Eco RI fragment of the P4 cDNA at the 5’ end was lost. Therefore, a new cDNA library was constructed, again from fat body RNA of larvae induced with bacteria, and screened with one of the original cDNA clones (pP4-1) as a probe. Clones hybridizing with P4 cDNA were very abundant, accounting for 8.7% of the library. Ten positive clones were isolated and analyzed by restriction mapping. Three of these (pP4-5, pP4-9.2, and pP4-10.1) contained an Eco RI fragment of 1,100 bp and an additional fragment of 240 bp, which was missing from clones isolated from the first library. Two additional groups of clones (pP4-7and pP4-1.1, pP49.1) had restriction maps different from pP4-10.1 and from each other. Preliminary sequencing and Southern blot analysis indicate that they are part of a gene family (data not shown). Clone pP4-10.1 was completely sequenced (Fig. 1)and contained a 1,365 bp insert with an open reading frame beginning with an ATG codon at position 41 and extending to position 1273, followed by a 73 bp 3’ untranslated sequence and a poly(A) tail, The open reading frame codes for a 411 residue polypeptide of M, = 45,678. The NH2-terminalsequence of the polypeptide is hydrophobic and likely encodes a signal peptide, responsible for secretion of the protein into the hemolymph. A predicted cotranslational cleavage site of the signal peptide [28] is after the 17th residue (Ala). Such a cleavage would result in a 394 residue polypeptide of Mr = 43,968. The sequence contains a putative N-linked glycosylation site (Asn Arg Thr) at position 265 of the mature protein. This is consistent with the estimation of 12% carbohydrate in P4 isolated from hemolymph and an observed M, of 48,000 [19]. The predicted NH2-terminalsequence was identical in only 11/18positions with the sequence previously determined by Edman degradation [ 191. The Edman degradation was repeated and yielded a sequence consistent with that predicted from the cDNA. We believe the differences result from errors in interpretation of the data reported previously. Protein P4 is a Member of the Ig Gene Superfamily

A search of the National Biomedical Research Foundation protein sequence data base (release 21.0) with the computer program FASTA f29j resulted in high scores with members of the Ig gene superfamily [30,31], particularly with several vertebrate cell adhesion molecules (optimized scores for mouse and chicken neural cell adhesion molecules were 259 and 186, respectively). Inspection of alignments of the M. sexta P4 and various Ig superfamily proteins showed that P4 contains four degenerate repeated sequences, each with pairs of cysteine residues that define Ig domains and other highly conserved residues typical of C2 (also called H) Ig domains 130,311 (Fig. 2). Because the members of the Ig gene superfamily are widely divergent and only a few residues near the cysteines are highly conserved, proteins in this superfamily can have less than 20%identical residues. Statistical comparisons

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A C O O T C C A G A T G T G T T C A A C A C A G C A A C A A C A C A G G T G A A M F K S I V A L A A C V A M C V A

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Fig. 1. cDNA and deduced amino acid sequence of M. sexta P4 (hemolin). Amino acids in the mature hemolyrnph protein are assigned positive numbers, and those in the putative signal peptide are assigned negative numbers. Amino acids which are underlined were confirmed by protein sequencing. Cysteines which define the four immunoglobulin domains are marked by A and a putative N-linked glycosylation site i s marked by *

are therefore important in determining whether a sequence is likely to be a superfamily member. The significance of the relationship of the four Ig-like domains of P4 to several vertebrate and Drosophila C2 Ig domains was evaluated with the ALIGN computer program [32], in which two sequences are given an alignment score and the sequences are then shuffled and realigned a num-

Manduca sexta Immunoglobulin-Related Protein

* P4-1 P4-2 P4-3

P4-4 NGLI FASC NCAM

VCAM TAGl alB

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G h f C P hWp P L f D G o C N ESQATVLECVTENGDKDVKYSWQKDGKEFKW QEHNIAQRKDEGSLVFLKPEAKDEGKYRCFAESAAGV EGSWLKLECSIPEGYPKPTIVWRKQLGEDESIADSILARRITQSPEGDLYFTSVEKEDVSESYKWCAAKSPAID AGDVTMIYCMYG GVPMAYPNWFKDGKDVNG KPSDRITRHNRTSGKRLLIKETLLEDRGTFTCDVNNEVGK KQARLVIPCEVS RYPAAPVSWTPNAKPISG RWVSPSGLTIKGIQKSDKGYYGCQAHNEHGD RGKRMELFCIYG GTPLPQTVWSKDCQRIQW SDRIT QGHYGKSLVIRQTNFDDAGTYTCDVSNGVGN EGTDFTAKCAAS GKPVPRYTWIRVDTARDL TKDGDRVSADVLLGELRIREVRPEDAANYSCTAKNAAGT LGQSVTLVCDAD GFPEPTMSWTKDGEPIEN EEEDDEKHIFSDDSSELTIRDKNDEAEYVCIAENKAGE EGGSVTMTCSSE GLPAPEIFWSKKLDNGNL QHLSGNATLTLIAMRMEDSGIWCEGVNLIGK VGQQVTLECFAF GNPVPRIKWRKVDGSLSP QWATAEPTIJ?IPSVSFEDEETYECEAENSKGR PGNKVTLTCVA PLSGVDFQLRRGEKELLV PRSSTSPDRIFFHLNAVALGDGGHYTCRYRLHDNQ A

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Fig. 2. Alignment of the four Ig domains of Manduca P4 (hemolin) with C2 domains from several members of the Ig gene superfamily. Alignment was performed by aligning the cysteines and highly conserved residues by eye and inserting a minimum number of gaps to account for the variable lengths of the central portion of the domains. P4-1, P4-2, P4-3, and P4-4 are the four lg domains of Manduca P4. NCLl is Ig domain 3 of Drosophila neuroglian [141. FASC is Ig domain 3 of Drosophila fasciclin [121. NCAM is Ig domain 3 of mouse neural cell adhesion molecule [41].VCAM is Ig domain 3 of human vascular cell adhesion molecule [42]. TAG-I i s Ig domain 3 of rat TAG1 [331. d B i s Ig domain 3 of human a,B-glycoprotein [36]. Cysteines are markedwith *. Residues present in at least 6 of the 10 sequences are markedwith A.Conserved residues in C2 ( = H) domains as described by Hunkapillar and Hood [311 are shown above the aligned sequences. Upper case letters are single-letter amino acid code. Lower case letters represent amino acids with similar physical properties: o-aromatic: Y,F,W; f-aliphatic: L,I,V; p-polar:K,R,H,D,E,Q,N,T,S; h-hydrophobic:L,I,V,M,Y,F.

ber of times, allowing comparison of the original alignment score with those generated from randomized sequences with the same amino acid compositions (Table 1). The comparisons reveal consistently high scores when comparing the domains of M. sextu P4 with C2 domains from several Ig superfamily members, with scores in the same range as for comparisons among the various vertebrate and Drosophila C2 sequences. The P4 sequence thus fits established criteria for inclusion in the Ig gene superfamily [31]: statistically significant sequence similarity to several representatives of the superfamily and the presence of the Set of highly conserved residues flanking the pair of cysteines in each domain. TABLE 1. Statistical Comparisonsof the Ig Domains in Manduca P4 With q p e C2 Ig Domains* P4-1 P4-1 P4-2 P4-3 P4-4

NGLI FASC NCAM VCAM alB

P4-2

P4-3

P4-4

NGLI

5.76

6.02 3.25

3.13 6.19 5.22

7.22 6.15 11.72 7.60

FASC NCAM VCAM 7.39 4.80 5.71 5.95 8.06

6.67 9.14 6.60 10.37 10.40 8.50

6.35 7.03 5.67 7.66 8.44 8.09 9.93

TAGl

alB

6.54 5.14 6.33 5.91 8.18 8.49 7.42 10.05

4.96 4.37 4.82 4.41 4.68 4.53 3.92 5.04 7.85

'Sequences compared and abbreviations are the same as those in Figure 2. Comparison scores determined by the ALIGN program [31] are expressed as the number of standard deviations above the mean score for shuffled sequences (bias = 6, gap penalty = 6, 100 random runs per comparison). Scores of 3.1, 4.3, and 5.2 standard deviations indicate chance probabilities of and loL7,respectively.

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MANDUCA

1 QPVEI(MPVLKDQPAEVLFRESQA~~C~E.NGDKMSISWQKDGKEFKWQEHNIAQRKD60

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121 LECSIPEGYPKPTIWVRKQLGEDESIAD.SILARR1TQSPEGDLYFTSVEKEDVSESYKY

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119 179 179

180 VCAAKSPAIDGDVPLVGYTIKSL~KNTNQKNGELVPMYVSNDMI~GDV~IYCMYGGV 239

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SN CECROPIA 180 V C T A K N A A V D E E W L V E Y E I K G ~ K D N S G Y K G E P V P Q Y V S K D ~ ~ D V ~ I Y C M Y G239 MANDUCA

240 PMAYPNWFKDGKDVNGKPSDRITRHNRTSGKRLLIKETLLIKETLLEDRGTFTCD~NEVGKPQKH 299

I t I l l I 1 IIIIII I I I I I I I I I I I I I I I I I I I I f I I I I I I I I I I I I

CECROPIA 240 PMGYPNYFKNGKDVNGNPEDRITRHNRTSGKRLLFKTTLPEDEGVYTCEVDNGVGKPQKH 299 MANDUCA

300 SVXLTWSGPRFTKKPEKQVIAKQAR.LVIPCEVSRYPAAPVSWTFNAKP1SGSRWASP

CECROPIA 300 MANDUCA

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359 SGLTIKGIQKSDKGYYGCQAHNEHGDAYAETLVIVA 394

I l l II

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Fig. 3. Alignment of the amino acid sequences of M. sexta and H. cecropia hemolins (P4) [181. The sequences were aligned with the GAP computer program [43] using gap weight = 3.0 and gap length weight = 0.1,

While this paper was in preparation, the cDNA and deduced amino acid sequence of H . cecropia P4, renamed hemolin, was published [18] and shown to be a member of the Ig superfamily. In an alignment of the M. sexta and H . cecropia P4 sequences, the two proteins contain 60%identical amino acids (Fig. 3). The putative N-linked glycosylation site at position 265 is conserved, although glycosylation of the H. cecropia protein has not been detected [20]. When the Ig domains (as defined in Fig. 2) from the two proteins are compared individually, domain 1 contains 68% identical residues (46/68); domain 2: 49% (37176);domain 3: 77% (54170); and domain 4: 64% (41/64). To promote consistency in nomenclature for these homologous proteins, we will hereafter refer to M. sexta P4 as "hemolin." Expression of the Hemolin Gene Induced by Bacteria Previous experiments have shown that the hemolymph concentration of hemolin increases 30-40 fold within 2 days after injection of Gram negative or Gram positive bacteria, and that isolated components of bacterial cell walls can also induce increased levels of hemolin in hemolymph [19]. To examine the early period of induction, hemolin mRNA from fat body was analyzed by Northern blotting after injection of both larval and adult insects with M . lysodeikticus cells (Fig. 4). Hemolin mRNA (1.4 kb) was not detectable in naive larvae, but appeared within 1 h after injection of bacteria and continued to increase in abundance until at least 24 h. No hybridization could be detected in RNA from larvae injected with water as a control. In adults, a 1.4 kb mRNA

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Fig. 4. Northern blot analysis of hemolin mRNA induction after injection of bacteria. Day 2 fifth instar larvae or day 1 adult males were injected with 100 pg of M. lysodeiktikus cells in 10 pl water or with 10 pI water alone (control). At various times after injection, fat body RNA was extracted, and samples (10 FLg total RNA) were analyzed by agarose gel electrophoresis in the presence of formaldehyde, transferred to nylon membrane, and hybridized with 32P labeled hemolin cDNA. Numbers above the lanes indicate hours after injection. Lanes marked C indicate RNA from control animals.

hybridizing with the hemolin clone was detectable at a low level in uninjected insects and did not increase in abundance until 4 h after injection of bacteria, then continued to accumulate until at least 24 h but did not reach the abundance achieved in larval fat body. A larger mRNA of 3 kb that hybridized with the hemolin cDNA was also induced by bacteria in both larvae and adults. The identity of this mRNA is not yet known but may correspond to one of the cDNA clones isolated which had restriction maps different from clone pP4-10.2. Binding of Hemolin to Bacteria Sun et al. [18] reported that hemolin from H . cecropia bound to the surface of E. coli cells in a complex with an unidentified 125,000 dalton hemolymph protein. We have performed a similar experiment to examine the association of M. sextu hemolymph proteins with bacteria. Hemolymph proteins from naive larvae and from larvae previously injected with M. lysodeikticus were incubated with E . coli cells for 1 min. The cells were then washed with water, and hemolymph proteins remaining bound were eluted with 0.5 M ammonium formate and analyzed by SDS polyacrylamide gel electrophoresis and immunoblotting (Fig. 5). The major hernolymph proteins eluted from the bacterial surface in the case of either naive or immunized larvae were the subunits of the lipid transport protein, lipophorin: apolipophorin-I (M, = 250,000) and apolipophorin-I1(M, = 80,000) [4].The identity of these bands visible on the

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Fig. 5. Association of hemolymph proteins with E. coli cells. Hemolymph proteins (200 11.1) from naive larvae or larvae injected 24 h previously with 100 p g M. lysodeiktikus were incubated with a suspension of E. coli cells (200 $1. In some cases bovine serum albumin was added to the hemolymph proteins at 10 mg/ml prior to incubation with bacteria. The cells were washed twice with water, and bound proteins were eluted with 200 pI of 0.5 M ammonium formate, pH 6.4. Samples of the total hemolymph proteins (2 11.1) and those eluted from the bacterial cells (30 pl) were analyzed by SDS polyacrylamide gel electrophoresis (10% acrylamide gel) followed by staining with Coomassie blue R (panel A) or irnmunoblotting and detection with antibody to hemolin (P4) (panel B). lane 1: Naive, total hemolymph proteins; lane 2: naive, eluted proteins; lane 3: naive f bovine serum albumin, total hemolymph proteins; lane 4: naive bovine serum albumin, eluted proteins; lane 5: immunized, total hemolymph proteins; lane 6 : immunized, eluted proteins; lane 7: immunized + bovine serum albumin, total hemolymph proteins; lane 8: immunized bovine serum albumin, eluted proteins.

+

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stained gel was confirmed with immunoblotting, using lipophorin antibodies (data not shown). The amount of lipophorin bound was not affected when a large excess of bovine serum albumin was present, indicating some specificity in the interaction of lipophorin with the bacteria, presumably an association of the lipoprotein with a hydrophobic surface. Apolipophorin-I11 (M, = 17,000), which associates with low density lipophorin, was also present in eluates from immunized larval hemolymph. It is known that in starving larvae, high density lipophorin is converted to a lower density lipophorin with associated apolipophorin-I11(K. Tsuchida and M. Wells, personal communication). We have observed that larvae injected with bacteria cease feeding for approximately 24 h, which may explain the presence of apolipophorin-I11in eluates from immunized, but not naive insects. At least one serine protease inhibitor from the serpin family [34] was also eluted from bacteria incubated

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with hemolymph proteins of naive or immunized larvae (also confirmed by immunoblotting-data not shown). There is some evidence for an association of serpins with lipophorin [35] which may explain this observation. Hemolin was present at a low level in hemolymph from naive larvae but could not be detected in the eluate from E. coli. When hemolymph from immunized larvae (in which hemolin is at much higher concentration [19])was tested, hemolin was present in the eluate from E. coli cells. The association of hemolin with the bacterial surface appeared to be slightly decreased when bovine serum albumin was added as a competitor. It should be noted that some hemolymph proteins, most obviously the very abundant storage proteins, were not observed to bind to bacteria, Hemolin Blocks Self Adhesion of Hernocytes In vertebrate immune systems, proteins with C2 type Ig domains act as leukocyte adhesion molecules, mediating cell-cell interactions during inflammation [15-171. A series of experiments was performed to test whether hemolin can affect aggregation of M. sexfu hemocytes. When hemocytes were placed in a plastic microtiter well, they began to aggregate, spread, and attach to the substrate (Fig. 6A). However, if hemolin was present at 160 pg/ml, about 1/10 the concentration reached in hemolymph after injection of bacteria [19], the hemocytes remained primarily as single, rounded cells, with very few small aggregates (Fig. 6B). When the same amount of ovalbumin, a glycoprotein similar in size to hemolin, was added as a control, the hemocytes aggregated in the same way as when buffer alone was added (Fig. 6C). Association of Hemolin With Hemocytes

One mechanism by which hemolin might inhibit aggregation is to bind to hernocyte surfaces, perhaps blocking a cell adhesion receptor. To test this, hemocytes attached to glass slides were incubated with hemolin and after washing,

Fig. 6. Effect of hemolin on hemocyte aggregation. Hemolymph from a Manduca fifth instar larva was mixed with an equal volume of 50 m M MOPS, 50 m M glutathione, pH 7 (A), 10 pg hemolin in the same buffer (B), or 10 pg ovalburnin in the same buffer (C) in the wells of a 96 well plate and incubated with shaking at room temperature for 3 h before phase contrast microscopy. Scale bar = 100 pm.

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Fig. 7. lrnrnunofluorescencestaining of Manduca hernocytes with hernolin antibody. A: Hernocytes treated with hemolin before washing and irnrnunofluorescence staining with rabbit antihernolin and rhodarnine-labeled goat anti-rabbit IgG. B: Hoechst (#33258) staining of the cells in A to show location of nuclei. C: Hernocytes untreated with hernolin stained as in A. D: Hoechst stained nuclei of cells shown in C. E: Hernocytes treated with hernolin as in A but using preirnrnune serum as a control in palce of antihernolin. F: Hoechst stained nuclei of cells in E. Scale bar = 10 prn.

associated hemolin was detected by immunofluorescence (Fig. 7). In the cells which were not treated with hemolin a low level of antibody binding could be detected (Fig. 7C)indicating that some hemolin may be present in association with hemocytes from naive larvae. When hemolin was added to the cells the amount of immunofluorescence was greatly increased (Fig. 7A). Even though the cells were extensively washed, hemolin appeared to remain bound to their surface. In a negative control, cells treated with hemolin did not give an immunofluorescence signal when preimmune serum was used instead of hemolin antiserum (Fig. 7E). Thus, the association of hemolin with hemocyte surfaces may be involved in the mechanism by which hemolin blocks hemocyte aggregation.

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DISCUSSION

We have isolated a cDNA clone for hemolin (P4),the major bacteria-induced protein in hemolymph of M. sextu [19]. The deduced amino acid sequence shows that hemolin is a member of the Ig gene superfamily. M. sexta hemolin is composed of four Ig domains of the C2 type [30] which are present in many cell adhesion molecules and are thought to represent more closely the prototypical Ig domain than the C and V domains characteristic of constant and variable regions of antibody molecules [31]. The three dimensional structure of Ig domains is consistent with the circular dichroism spectrum of hemolin from H. cecropia, which indicates that hemolin is predominantly composed of p sheet [20]. Most proteins containing C2 domains exist primarily as membrane proteins, although some also exist in soluble forms [31,33]. However, hemolin does not contain regions likely to be transmembrane or phosphatidylinositollinked membrane attachment sequences. Hemolin bears an overall structural resemblance to al13-glycoprotein [36] from human plasma, which is a soluble protein composed of five Ig domains and whose physiological function is unknown, The amino acid sequences of the Ig domains in hemolin are, however, more closely related to the domains of several cell adhesion proteins than to those of alB-glycoprotein (Table 1). The M. sexta hemolin amino acid sequence is identical in 60% of its residues with hemolin from H. cecropia. The most highly conserved of the four Ig domains is domain 3, which contains 77% identical residues, perhaps indicating a significant role for this domain in the function of the protein. Hemolin is present at a low level in naive M. sexfa larvae (insects reared with precautions taken to minimize exposure to bacteria) and is highly induced after injection of Gram positive or Gram negative bacteria or the bacterial cell surface components, peptidoglycan and lipopolysaccharide [191. Increased levels of hemolin mRNA in fat body cart be detected within 1h after injection of bacteria (Fig. 4)and elevated hemolin protein can be detected in hemolymph by Western blotting by 4 h (data not shown). In hemolymph of Manduca adults a protein antigenically related to hemolin, postlarval protein, is present at a low level in the absence of infection [19,37]. RNA from naive adults which hybridizes with the hemolin clone (Fig. 4)may be postlarval protein mRNA. Induction of hemolin mRNA is slower in adults than in larvae, with increased levels detectable only by 4 h after injection of bacteria. Hemolin appears to associate with the surface of bacterial cells. Sun et al. [18] found that hemolin from H. cecropia pupae bound to E . coli cells as a complex with a 125,000 dalton hemolymph protein. In this study we found that hemolin from immunized M. sexta larvae associated with the surface of E. coIi cells, but we did not observe binding of a 125,000 dalton protein. Another difference was the finding that lipophorin and serpins from M. sexta associated with E . coli cells, which was not observed with H . cecropia [MI.Some differences in the two experiments might contribute to the different results, Sun et al. [18] used an ammonium sulfate fraction as a source of hemolymph proteins, which may have lead to the loss of lipophorin and serpins from their sample, whereas in this study total hemolymph proteins were used in the experiments. In addition, there may be differences between the experiments as a result of comparing pupal hemolymph [18] with larval hernolymph. For

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example, in M. sextu pupae there is a constitutively expressed protein related, but not identical, to hemolin [19]. Nevertheless, consistent between the two studies is the finding that hemolin, but not most other hemolymph proteins, binds to the surface of bacteria. The ability of hemolin to bind to bacteria and to hemocytes suggests a possible role as an opsonin, a speculation which has not yet been tested, The similarity of the Ig domains in hemolin to those from various cell adhesion molecules suggests that hemolin may have a role in regulation of cell adhesion during the cellular responses to bacterial infections. Experiments from this study (Fig. 7) and others 120,381 demonstrate that hemolin can associate with hemocyte surfaces. The presence of hemolin appears to inhibit the selfassociation of hemocytes and adhesion of hemocytes to surfaces (Fig. 6) [38]. The in vivo function of this activity during a bacterial infection of the insect is unclear. The protein might cause release of sessile hemocytes from places of attachment on the body wall to increase the number of hemocytes in circulation available for fighting an infection [27]. Another hypothetical function of hemolin may be to prevent excessive aggregation of hemocytes into nodules large enough to interfere with circulation or to prevent all of the hemocytes from being used in nodule formation, keeping a population in reserve for subsequent infections. Interestingly, a hymenopteran parasitoid of lepidopteran larvae covers its eggs with particles that contain a protein related to hemolin, which blocks encapsulation of the egg by the host's hemocytes [38,39]. The relevance of invertebrate recognition and defense mechanisms as model systems and with regard to evolution of the immune response has been debated [40]. The discovery that bacterial infection in an insect induces synthesis of a protein from the Ig gene superfamily that affects interactions of blood cells points to a possible common ancestry of the mechanisms by which vertebrates and invertebrates recognize and respond to foreignness. Changes in the adhesive properties of invertebrate hemocytes and vertebrate leukocytes appear to be hallmarks of the initial responses to infection, and in vertebrates these changes are mediated by cell adhesion proteins containing Ig domains [16,171. Preliminary evidence indicates that hemolin is part of a gene family in M. sextu. A 3 kb mRNA, in addition to the 1.4 kb mRNA which apparently encodes the hemolin sequence, is induced by bacteria and hybridizes with the hemolin cDNA (Fig. 3). Also, two classes of cDNA clones which are related in sequence to hemolin have been isolated, and two hemolymph proteins, postlarval protein [19,37] and a second, less abundant induced protein from larvae (Ladendorff and Kanost, unpublished) appear to be related but not identical to hemolin. The identification of hemolin as a member of the Ig gene superfamily, with effects on adhesive properties of hemocytes suggests that the phenomena of recruitment and response to infection by invertebrate hemocytes may not be as different from those of vertebrate blood cells as previously supposed. LITERATURE CITED 1. Dunn PE: Biochemical aspects of insect immunology. Annu Rev Entomol32,321(1986). 2. Boman HG, Hultmark D: Cell-free immunity in insects. Annu Rev Microbiol41,103 (1987). 3. Lackie AM: Immune mechanisms in insects. Parasitol Today 4,98 (1988).

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4. Kanost MR, Kawooya JK, Law JH, Ryan RO, Van Heusden MC, Ziegler R: Insect haemolymph proteins. Adv Insect Physiol22,299 (1990). 5. Matsumoto N, Okada M, Takahashi€3, Qu X-M, Nakajima Y, Nakanishi Y, Komano H, Natori S: Molecular cloning of a cDNA and assignment of the C-terminal of sarcotoxin IA, a potent antibacterial protein of Sarcophuga peregrina. Biochem J 239, 717 (1986). 6. Ando K, Natori S Molecular cloning, sequencing, and characterization of cDNA for sarcotoxin IIA, an inducible antibacterial protein of Sarcophuga peregrina (flesh fly). Biochemistry 27, 1715 (1988). 7. Dickinson L, Russell V, Dunn PE: A family of bacteria-regulated, cecropin D-like peptides fromManduca sexta. J Biol Chem 263,19424 (1988). 8. Kylsten P, Samakwlis C, Hultmark D: The cecropin locus in Drosophila:a compact gene cluster involved in the response to infection. EMBO J 9,217 (1990). 9. Faye I, Pye A, Rasmuson T, Boman HG, Boman IA: Insect immunity 11. Simultaneous induction of antibacterial activity and selective synthesis of some hemoIymph proteins in diapausing pupae of Hyulophoru cecropia and Samia cynfhia. Infect Immun 12,1426 (1975). 10. Kanost MR, Dai W, Dunn PE: Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta. Arch Insect Biochem Physiol 8,147 (1988). 11. Marchalonis JJ, Schluter SF: Immune-like recognition molecules of invertebrates and primitive vertebrates. Bioscience 40, 758 (1990). 12. Harrelson AL, Goodman CS: Growth cone guidance in insects: Fasciclin I1 is a member of the immunoglobulin superfamily. Science 242,700 (1988). 13. Seeger MA, Haffley L, Kaufman TC: Characterization of amalgam: a member of the immunoglobulin superfamily from Drosophila. Cell 55, 589 (1988). 14. Bieber AJ, Snow PM, Hartsch M, Pate1 NH, Jacobs JR, Traquina ZR, Schilling J, Goodman CS: Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1.Cell 59,447 (1989). 15. Albelda SM, Buck CA: Integrins and other ceIl adhesion molecules. FASEB J 4,2868 (1990). 16. Osborn L: Leukocyte adhesion to endothelium in inflammation. Cell 62,3 (1990). 17. Springer TA: Adhesion receptors of the immune system, Science 346,425 (1990). 18. Sun S-C, Lindstrom I, Boman HG, Faye 1, Schmidt 0: Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily. Science 250,1729 (1990). 19. Ladendorff NE, Kanost MR: Isolation and characterization of bacteria-induced protein P4 from hemolymph of Manduca sexfu. Arch Insect Biochem Physiol15,33 (1990). 20. Andersson K, Steiner H: Structure and properties of protein P4, the major bacteria-inducible protein in pupae of Hyalophora cecropia. Insect Biochem 17, 133 (1987). 21. Cole KD, Fernando-Warnakulasuria GJF', Boguski MS, Freeman M, Gordon JI, Clark WA, Law JH, Wells MA: Primary structure and comparative sequence analysis of an insect apolipoprotein. J Biol Chem 262,11794 (1987). 22. Chinzei Y, White BN, Wyatt GR: Vitellogenin mRNA in locust fat body: identification, isolation and quantitative changes induced by juvenile hormone. Can J Biochem 60,243 (1982). 23. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982). 24. Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28,351 (1984). 25. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74,5463 (1977). 26. Tabor S, Richardson CC: DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84,4767 (1987). 27. Horohov DW, Dunn PE: Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria. J Invertebr Path01 40,327 (1982). 28. von Heijne G : A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14,4683 (1986). 29. Pearson WR, Liprnan DJ: Improved tools for biological sequence analysis. Proc Natl Acad Sci USA 85, 2444 (1988). 30. Williams AF, Barclay AN: The immunoglobulin superfamiIy-domains for cell surface recognition. Annu Rev Immunol6,381(1988). 31. Hunkapiller T, Hood L: Diversity of the immunoglobulin gene superfamily. Adv Immunol 44, l(1989).

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32. Dayhoff MO, Barker WC, Hunt LT: Establishing homologies in protein sequences. Methods Enzymol92,524 (1983). 33. Furley AJ, Morton SB, Manalo D, Karagogeos D, Dodd J, Jessell TM: The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity. Cell 62,157 (1990). 34. Kanost MR: Isolation and characterization of four serine proteinase inhibitors (serpins) from hernolymph of Munduca sexta. Insect Biochem 20,141 (1990). 35. Kanost MR, Prasad SV, Wells MA: Primary structure of a member of the serpin superfamily of proteinase inhibitors from an insect, Munduca sexta. J Biol Chem 264,965 (1989). 36. Ishioka N, Takahashi N, Putnam Fw:Amino acid sequence of human plasma a,B-&coprotein: homology to the immunoglobulin supergene family. Proc Natl Acad Sci USA 83,2263 (1986). 37. Ryan RO, Cole KD, Kawooya JK, Wells MA, Law JH: Identification and characterization of a novel postlarval hernolymph protein from Manduca sextu. Arch Insect Biochem Physiol9, 81 (1988). 38. Schmidt 0, Anderson K, Will A, Schuchmann-Feddersen I: Viruslike particle proteins from a hymenopteran endoparasitoid are related to a protein component of the immune system in the lepidopteran host. Arch Insect Biochem PhysioIZ3, 107 (1990). 39. Berg R, Schuchmann-Feddersen I, Schmidt 0:Bacterial infection induces a moth (Ephestiu kuhniella) protein which has antigenic similarity to virus-like particle proteins of a parasitoid wasp (Venturia cunescens). J Insect Physiol34,473 (1988). 40. Marchalonis JJ, Schluter SF: On the relevance of invertebrate recognition and defence mechanisms to the emergence of the immune response of vertebrates. Scand J Immunol32, 13 (1990). 41. Barthels D, Santoni J-J, Willie W, Ruppert C, Chaix J-C, Hirsch M-R, Fontecilla-Camps JL, Goridis C: Isolation and nucleotide sequence of mouse NCAM cDNA that codes for a M, 79,000 polypeptide without a membrane-spanning region. EMBO J 6,907 (1987). 42. Osborn L, Hession C , Tizard R, Vassallo C, Luhowshyj S, Chi-Ross0 G, Lobb R Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59, 1203 (1989). 43. Devereux J, Haeberli , ' l Smithies 0:A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12,387 (1984).

Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larv...
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